Human protease associated proteins

ABSTRACT

The invention provides human protease associated proteins (HPRAP) and polynucleotides which identify and encode HPRAP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of HPRAP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of human protease associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and immune disorders.

BACKGROUND OF THE INVENTION

Proteolytic processing is an essential component of normal cell growth, differentiation, remodeling, and homeostasis. The cleavage of peptide bonds within cells is necessary for a variety of functions, including the maturation of precursor proteins to their active form, the removal of signal sequences from targeted proteins, the degradation of incorrectly folded proteins, and the controlled turnover of peptides within the cell. Proteases participate in apoptosis, inflammation, and in tissue remodeling during embryonic development, wound healing, and normal growth. They are necessary components of bacterial, parasitic, and viral invasion and replication within a host. Mammalian proteases have been identified and categorized based on active site structure, mechanism of action, and overall three-dimensional structure. (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: A Practical Approach. Oxford University Press, New York, N.Y., pp. 1-5.)

The serine proteases (SPs) are a large family of proteolytic enzymes that include the digestive enzymes, trypsin and chymotrypsin; components of the complement and blood-clotting cascades; and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. SPs are named because of the presence of a serine residue found in the active catalytic site which forms a triad together with an aspartate and a histidine residue. SPs have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. The main sub-families are trypases, which cleave after arginine or lysine; aspases, which cleave after aspartate; chymases, which cleave after phenylalanine or leucine; metases which cleave after methionine; and serases, which cleave after serine.

Cysteine proteases are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Mammalian cysteine proteases include lysosomal cathepsins and cytosolic calcium activated proteases, calpains. Cysteine proteases are produced by monocytes, macrophages and other cells of the immune system which migrate to sites of inflammation and secrete various molecules necessary for the repair of damaged tissue. These cells may overproduce the same molecules and cause tissue destruction in certain disorders. For example, in autoimmune diseases such as rheumatoid arthritis, the secretion of the cysteine protease, cathepsin C, degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones.

Carboxypeptidases A and B are the principal mammalian representatives of the metallo-protease family. Both are exopeptidases of similar structure and active sites. Carboxypeptidase A, like chymotrypsin, prefers C-terminal aromatic and aliphatic side chains of hydrophobic nature, whereas carboxypeptidase B is directed toward basic arginine and lysine residues. Active site components include zinc, with its three ligands of two glutamic acid and one histidine residues.

Many other proteolytic enzymes do not fit any of the major categories discussed above because their mechanisms of action and/or active sites have not been elucidated. These include the aminopeptidases and signal peptidases. Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Bovine leucine aminopeptidase is a zinc metallo-enzyme that utilizes the sulfhydryl groups from at least three reactive cysteine residues at its active site in the binding of metal ions. (Cuypers, H. T. et al. (1982) J. Biol. Chem. 257:7086-7091.)

Signal peptidases are a specialized class of proteases found in all prokaryotic and eukaryotic cell types that serve in the processing of signal peptides from certain proteins. Signal peptides are amino-terminal sequences on a protein which directs the protein from its ribosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein. Signal peptidases exist as multi-subunit complexes in both yeast and mammals.

Proteasome, an intracellular protease complex found in some bacteria and in all eukaryotic cells, plays an important role in cellular physiology. Proteasomes are responsible for the timely degradation of cellular proteins of all types and control proteins that function to activate or repress cellular processes such as transcription and cell cycle progression. (Ciechanover, A. (1994) Cell 79:13-21.) Proteasomes act on proteins which have been targeted for hydrolysis by the covalent attachment of a small protein called ubiquitin to lysine side chains of the protein. Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins. (Ciechanover, supra.) Proteasomes are large (˜2000 kDa), multisubunit complexes composed of a central catalytic core containing a variety of proteases and terminal subunits that serve in substrate recognition and regulation of proteasome activity.

Protease inhibitors play a major role in the regulation of the activity and effect of proteases. They have been shown to control pathogenesis in animal models of proteolytic disorders. (Murphy, G. (1991) Agents Actions Suppl 35:69-76.) In particular, low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, seem to be correlated with malignant progression of tumors. (Calkins, C. et al (1995) Biol Biochem Hoppe Seyler 376:71-80.) The balance between levels of cysteine proteases and their inhibitors is also significant in the development of disorders. Specifically, increases in cysteine protease levels, when accompanied by reductions in inhibitor activity, are correlated with increased malignant properties of tumor cells and the pathology of arthritis and immunological diseases in humans.

The Kunitz family of serine protease inhibitors are characterized by one or more “Kunitz domains” containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds. Members of this family include aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), and inter-α-trypsin inhibitor, and bikunin. (Marlor, C. W. et al. (1997) J. Biol. Chem. 272:12202-12208.) Members of this family are potent inhibitors (in the nanomolar range) against serine proteases such as kallikrein and plasmin. Aprotinin has clinical utility in reduction of perioperative blood loss.

The discovery of new human protease associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cell proliferative and immune disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, human protease associated proteins, referred to collectively as “HPRAP” and individually as “HPRAP-1”, “HPRAP-2”, “HPRAP-3”, and “HPRAP-4.” In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.

The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, or to a fragment of any of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.

Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.

The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, a fragment of SEQ ID NO:7, and a fragment of SEQ ID NO:8. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, a fragment of SEQ ID NO:7, and a fragment of SEQ ID NO:8, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, a fragment of SEQ ID NO:7, and a fragment of SEQ ID NO:8.

The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4. In another aspect, the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4 in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4, as well as a purified agonist and a purified antagonist to the polypeptide.

The invention also provides a method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.

The invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4.

The invention also provides a method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2.

The invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2.

The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, and a fragment of SEQ ID NO:4 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E and 1F show the amino acid sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID NO:5) of HPRAP-1. The alignment was produced using MACDNASIS PRO software (Hitachi Software Engineering Co. Ltd., San Bruno, Calif.).

FIGS. 2A, 2B, 2C, 2D, and 2E, show the amino acid sequence (SEQ ID NO:2) and nucleic acid sequence (SEQ ID NO:6) of HPRAP-2. The alignment was produced using MACDNASIS PRO software.

FIGS. 3A, 3B, and 3C, show the amino acid sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:7) of HPRAP-3. The alignment was produced using MACDNASIS PRO software.

FIGS. 4A, 4B, 4C, 4D, and 4E, show the amino acid sequence (SEQ ID NO:4) and nucleic acid sequence (SEQ ID NO:8) of HPRAP-4. The alignment was produced using MACDNASIS PRO software.

FIGS. 5A, 5B, 5C, and 5D show the amino acid sequence alignments between HPRAP-1 (031381; SEQ ID NO:1) and an endooligopeptidase A related protein from Oryctolagus cuniculus (GI 2827886; SEQ ID NO:9), produced using the multisequence alignment program of LASERGENE software (DNASTAR Inc, Madison Wis.).

FIGS. 6A and 6B show the amino acid sequence alignments between HPRAP-2 (1319265; SEQ ID NO:2) and the kunitz type protease inhibitor, bikunin, from human (GI 2065529; SEQ ID NO:10), produced using the multisequence alignment program of LASERGENE software.

FIGS. 7A and 7B show the amino acid sequence alignments between HPRAP-3 (2057812; SEQ ID NO:3) and the human proteasome subunit, p27 (GI 2055256; SEQ ID NO: 11), produced using the multisequence alignment program of LASERGENE software.

FIGS. 8A, 8B, and 8C show the amino acid sequence alignments between HPRAP4 (529706; SEQ ID NO:4) and a vacuolar aminopeptidase-related protein from Caenorhabditis elegans (GI 529706; SEQ ID NO:12), produced using the multisequence alignment program of LASERGENE software.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS

“HPRAP,” as used herein, refers to the amino acid sequences of substantially purified HPRAP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, when bound to HPRAP, increases or prolongs the duration of the effect of HPRAP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HPRAP.

An “allelic variant,” as this term is used herein, is an alternative form of the gene encoding HPRAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding HPRAP, as described herein, include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide encoding HPRAP or a polypeptide with at least one functional characteristic of HPRAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HPRAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HPRAP. The encoded protein may also be “altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HPRAP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of HPRAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. In this context, “fragments,” “immunogenic fragments,” or “antigenic fragments” refer to fragments of HPRAP which are preferably about 5 to about 15 amino acids in length, most preferably 14 amino acids, and which retain some biological activity or immunological activity of HPRAP. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

“Amplification,” as used herein, relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., pp. 1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which, when bound to HPRAP, decreases the amount or the duration of the effect of the biological or immunological activity of HPRAP. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of HPRAP.

As used herein, the term “antibody” refers to intact molecules as well as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind HPRAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

The term “antigenic determinant,” as used herein, refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The term “antisense,” as used herein, refers to any composition containing a nucleic acid sequence which is complementary to the “sense” strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.

As used herein, the term “biologically active,” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” refers to the capability of the natural, recombinant, or synthetic HPRAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A.” Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation, an aqueous solution, or a sterile composition. Compositions comprising polynucleotide sequences encoding HPRAP or fragments of HPRAP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts, e.g., NaCl, detergents, e.g.,sodium dodecyl sulfate (SDS), and other components, e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

“Consensus sequence,” as used herein, refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCR Kit (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′ direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison, Wis.). Some sequences have been both extended and assembled to produce the consensus sequence.

As used herein, the term “correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding HPRAP, by Northern analysis is indicative of the presence of nucleic acids encoding HPRAP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding HPRAP.

A “deletion,” as the term is used herein, refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

The term “similarity,” as used herein, refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.” A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Inc., Madison Wis.). The MEGALIGN program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

As used herein, the term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C₀t or R₀t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

The words “insertion” or “addition,” as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.

The term “microarray,” as used herein, refers to an arrangement of distinct polynucleotides arrayed on a substrate, e.g., paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.

The terms “element” or “array element” as used herein in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.

The term “modulate,” as it appears herein, refers to a change in the activity of HPRAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HPRAP.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In this context, “fragments” refers to those nucleic acid sequences which, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.

The terms “operably associated” or “operably linked,” as used herein, refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.

The term “oligonucleotide,” as used herein, refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. As used herein, the term “oligonucleotide” is substantially equivalent to the terms “amplimer,” “primer,” “oligomer,” and “probe,” as these terms are commonly defined in the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. A biological sample suspected of containing nucleic acids encoding HPRAP, or fragments thereof, or HPRAP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solid support; a tissue; a tissue print; etc.

As used herein, the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope “A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent (e.g., formamide), temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25° C., more preferably of at least about 42° C., and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.

The term “substantially purified,” as used herein, refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

“Transformation,” as defined herein, describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed” cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

A “variant” of HPRAP, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

THE INVENTION

The invention is based on the discovery of new human protease associated proteins (HPRAP), the polynucleotides encoding HPRAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative and immune disorders.

Nucleic acids encoding the HPRAP-1 of the present invention were first identified in Incyte Clone 031381 from the promonocyte cell line cDNA library (THP1NOB01) using a computer search, e.g., BLAST, for amino acid sequence alignments. A consensus sequence, SEQ ID NO:5, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 031381 (THP1NOB01), 3618224 (EPIPNOT01), 991115 (COLNNOT11), 1223519 (COLNTUT02), 1723492 (BLADNOT06), 599353 (BRSTNOT02), 2132359 (OVARNOT03), 853456 (NGANNOT01), and 673630 (CRBLNOT01).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C, 1D, 1E, and 1F. HPRAP-1 is 460 amino acids in length and has a potential N-glycosylation site at residue N299, and potential phosphorylation sites for cAMP- and cGMP-dependent protein kinase at T132, for casein kinase II at S11, S29, S95, S135, S155, and S310, for protein kinase C at S11, S23, S90, T191, S231, S247, S282, S331, S344, and S404, and for tyrosine kinase at Y87. As shown in FIGS. 5A, 5B, 5C, and 5D, HPRAP-1 has chemical and structural similarity with an endooligopeptidase A related protein from O. cuniculus (GI 2827886; SEQ ID NO:9). In particular, HPRAP-1 and the endooligopeptidase A related protein share 89% identity, including the potential N-glycosylation site and most of the potential phosphorylation sites found in HPRAP-1. The fragment of SEQ ID NO:5 from about nucleotide 1152 to about nucleotide 1205 is useful, for example, as a hybridization probe. Northern analysis shows the expression of this sequence in various libraries, at least 48% of which are immortalized or cancerous and at least 36% of which involve immune response. Of particular note is the expression of HPRAP-1 in cardiovascular and gastrointestinal tissues.

Nucleic acids encoding the HPRAP-2 of the present invention were first identified in Incyte Clone 1319265 from the bladder tissue cDNA library (BLADNOT04) using a computer search, e.g., BLAST, for amino acid sequence alignments. A consensus sequence, SEQ ID NO:6, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 1319265 (BLADNOT04), 1522685 (BLADTUT04), 872599 and 875890 (LUNGAST01), 1427703 (SINTBST01), 1224264 (COLNTUT02), and 1493438 (PROSNON01).

In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:2, as shown in FIGS. 2A, 2B, 2C, 2D, 2E. HPRAP-2 is 349 amino acids in length and has two potential N-glycosylation sites at residues N53 and N342, and potential phosphorylation sites for casein kinase II at T43, S169, S193, S264, S331, and T332, and for protein kinase C at S60, S71, S205, S259, S264, and T344. HPRAP-2 contains a potential signal peptide sequence between residues M1 and P48 and a potential transmembrane domain between residues M285 and C305. HPRAP-2 also contains two sequences related to the Kunitz family domains between residues C94 and C119, and residues C235 and C260. As shown in FIGS. 6A and 6B, HPRAP-2 has chemical and structural similarity with the kunitz type protease inhibitor, bikunin, from human (GI 2065529; SEQ ID NO:10). In particular, HPRAP-2 and human bikunin share 25% identity, including 12 cysteine residues located in the two kunitz domains in both proteins. The fragment of SEQ ID NO:6 from about nucleotide 500 to about nucleotide 563 is useful, for example, as a hybridization probe. Northern analysis shows the expression of this sequence in various libraries, at least 60% of which are immortalized or cancerous and at least 41% of which involve immune response. Of particular note is the expression of HPRAP-2 in reproductive and gastrointestinal tissues.

Nucleic acids encoding the HPRAP-3 of the present invention were first identified in Incyte Clone 2057812 from the bronchial epithelium cDNA library (BEPINOT01) using a computer search, e.g., BLAST, for amino acid sequence alignments. A consensus sequence, SEQ ID NO:7, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2057812 (BEPINOT01), 2496092 (ADRETUT05), and 026889 (SPLNFET01).

In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 3A, 3B, and 3C. HPRAP-3 is 239 amino acids in length and has a potential N-glycosylation site at residue N204, and potential phosphorylation sites for casein kinase II at S2, T19, and S65, and for protein kinase C at S46, T74, and S121. As shown in FIGS. 7A and 7B, HPRAP-3 has chemical and structural similarity with the human proteasome subunit, p27 (GI 2055256; SEQ ID NO:11). In particular, HPRAP-3 and p27 share 87% identity. The fragment of SEQ ID NO:7 from about nucleotide 551 to about nucleotide 604 is useful, for example, as a hybridization probe. Northern analysis shows the expression of this sequence in various libraries, at least 51 % of which are immortalized or cancerous and at least 36% of which involve immune response. Of particular note is the expression of HPRAP-3 in cardiovascular, hematopoietic/immune, nervous, and reproductive tissues.

Nucleic acids encoding the HPRAP-4 of the present invention were first identified in Incyte Clone 2058485 from the ovarian tissue cDNA library (OVARNOT03) using a computer search, e.g., BLAST, for amino acid sequence alignments. A consensus sequence, SEQ ID NO:8, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2058485 (OVARNOT03), 3115916 (LUNGTUT13), 556715 (MPHGLPT02), 2023129 (CONNNOT01), 1513114 (PANCTUT01), 2023129 (CONNNOT01), and 1964262 (BRSTNOT04).

In another embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:4, as shown in FIGS. 4A, 4B, 4C, 4D, and 4E. HPRAP-4 is 471 amino acids in length and has a potential N-glycosylation site at residue N65, and potential phosphorylation sites for cAMP- and cGMP-dependent protein kinase at S102 and S320, for casein kinase II at T124, S227, T292, T327, and S437, and for protein kinase C at T49, S102, S143, S227, and S437. As shown in FIGS. 8A, 8B, and 8C, HPRAP-4 shares chemical and structural similarity with a vacuolar aminopeptidase-related protein from C. elegans (GI 529706; SEQ ID NO:12). In particular, HPRAP-4 and the vacuolar aminopeptidase-related protein share 46% identity. Fragments of SEQ ID NO:8 from about nucleotide 254 to about nucleotide 319, and from about nucleotide 1025 to about nucleotide 1097 are useful, for example, as hybridization probes. Northern analysis shows the expression of this sequence in various libraries, at least 50% of which are immortalized or cancerous and at least 32% of which involve immune response. Of particular note is the expression of HPRAP-4 in reproductive and gastrointestinal tissues.

The invention also encompasses HPRAP variants. A preferred HPRAP variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the HPRAP amino acid sequence, and which contains at least one functional or structural characteristic of HPRAP.

The invention also encompasses polynucleotides which encode HPRAP. In a particular embodiment, the invention encompasses a polynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, which encodes an HPRAP.

The invention also encompasses a variant of a polynucleotide sequence encoding HPRAP. In particular, such a variant polynucleotide sequence will have at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HPRAP. A particular aspect of the invention encompasses a variant of SEQ ID NO:2 which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HPRAP.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding HPRAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HPRAP, and all such variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HPRAP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HPRAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HPRAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding HPRAP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encode HPRAP and HPRAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HPRAP or any fragment thereof.

Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, a fragment of SEQ ID NO:7, and a fragment of SEQ ID NO:8 under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.)

Methods for DNA sequencing are well known and generally available in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASET amplification system (GIBCO BRL, Gaithersburg, Md.). Preferably, the process is automated with machines such as the MICROLAB 2200 system (Hamilton, Reno, Nev.), DNA ENGINE thermal cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI Catalyst and ABI PRISM 373 and 377 sequencing systems (Perkin Elmer).

The nucleic acid sequences encoding HPRAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto, Calif.) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences Inc., Plymouth, Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68° C. to 72° C.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5′ regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode HPRAP may be cloned in recombinant DNA molecules that direct expression of HPRAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express HPRAP.

The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HPRAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

In another embodiment, sequences encoding HPRAP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Symp. Ser. (7):215-223, and Horn, T. et al. (1980) Nucl. Acids Symp. Ser. (7):225-232.) Alternatively, HPRAP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Perkin Elmer). Additionally, the amino acid sequence of HPRAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman and Co., New York, N.Y.)

In order to express a biologically active HPRAP, the nucleotide sequences encoding HPRAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ untranslated regions in the vector and in polynucleotide sequences encoding HPRAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HPRAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding HPRAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HPRAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and periodic supplements) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to contain and express sequences encoding HPRAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HPRAP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding HPRAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene) or PSPORT1 plasmid (Life Technologies, Gaithersburg Md.). Ligation of sequences encoding HPRAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of HPRAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of HPRAP may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of HPRAP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) Methods Enzymol. 153:516-54; Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)

Plant systems may also be used for expression of HPRAP. Transcription of sequences encoding HPRAP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding HPRAP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses HPRAP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.

For long term production of recombinant proteins in mammalian systems, stable expression of HPRAP in cell lines is preferred. For example, sequences encoding HPRAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Nati. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.), β glucuronidase and its substrate β-D-glucuronoside, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding HPRAP is inserted within a marker gene sequence, transformed cells containing sequences encoding HPRAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HPRAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encoding HPRAP and that express HPRAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of HPRAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HPRAP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et al. (1997 and periodic supplements) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; and Maddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HPRAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding HPRAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech (Piscataway, N.J.), Promega (Madison, Wis.). Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HPRAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode HPRAP may be designed to contain signal sequences which direct secretion of HPRAP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding HPRAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric HPRAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HPRAP activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the HPRAP encoding sequence and the heterologous protein sequence, so that HPRAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeled HPRAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega, Madison, Wis.). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably ³⁵S-methionine.

Fragments of HPRAP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431A peptide synthesizer (Perkin Elmer). Various fragments of HPRAP may be synthesized separately and then combined to produce the full length molecule.

THERAPEUTICS

Chemical and structural similarity exists between HPRAP-1 and an endooligopeptidase A related protein from O. cuniculus (GI 2827886). In addition, HPRAP-1 is expressed in cancer and immortalized cell lines and tissues associated with inflammation and the immune response. Therefore, HPRAP-1 appears to play a role in cell proliferative and immune disorders.

Chemical and structural similarity exists between HPRAP-2 and the kunitz type protease inhibitor, bikunin, from human (GI 2065529). In addition, HPRAP-2 is expressed in cancer and immortalized cell lines and tissues associated with inflammation and the immune response. Therefore, HPRAP-2 appears to play a role in cell proliferative and immune disorders.

Chemical and structural similarity exists between HPRAP-3 and the human proteasome subunit, p27 (GI 2055256). In addition, HPRAP-3 is expressed in cancer and immortalized cell lines and tissues associated with inflammation and the immune response. Therefore, HPRAP-3 appears to play a role in cell proliferative and immune disorders.

Chemical and structural similarity exists between HPRAP-4 and a vacuolar aminopeptidase-related protein from C. elegans (GI 529706). In addition, HPRAP-4 is expressed in cancer and immortalized cell lines and tissues associated with inflammation and the immune response. Therefore, HPRAP-4 appears to play a role in cell proliferative and immune disorders.

Therefore, in one embodiment, HPRAP-2 or a fragment or derivative thereof may be administered to a subject to treat or prevent a cell proliferative disorder. Such disorders can include, but are not limited to, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

In another embodiment, a vector capable of expressing HPRAP-2 or a fragment or derivative thereof may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising a substantially purified HPRAP-2 in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity of HPRAP-2 may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those listed above.

In another embodiment, HPRAP-2 or a fragment or derivative thereof may be administered to a subject to treat or prevent an immune disorder. Such disorders can include, but are not limited to, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.

In another embodiment, a vector capable of expressing HPRAP-2 or a fragment or derivative thereof may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising a substantially purified HPRAP-2 in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity of HPRAP-2 may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those listed above.

In a further embodiment, an antagonist of HPRAP-1 may be administered to a subject to treat or prevent a cell proliferative disorder. Such a disorder may include, but is not limited to, those discussed above. In one aspect, an antibody which specifically binds HPRAP-1 may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HPRAP-1.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HPRAP-1 may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.

In a further embodiment, an antagonist of HPRAP-3 may be administered to a subject to treat or prevent a cell proliferative disorder. Such a disorder may include, but is not limited to, those discussed above. In one aspect, an antibody which specifically binds HPRAP-3 may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HPRAP-3.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HPRAP-3 may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.

In a further embodiment, an antagonist of HPRAP-4 may be administered to a subject to treat or prevent a cell proliferative disorder. Such a disorder may include, but is not limited to, those discussed above. In one aspect, an antibody which specifically binds HPRAP-4 may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HPRAP-4.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HPRAP-4 may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.

In a further embodiment, an antagonist of HPRAP-1 may be administered to a subject to treat or prevent an immune disorder. Such a disorder may include, but is not limited to, those discussed above. In one aspect, an antibody which specifically binds HPRAP-1 may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HPRAP-1.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HPRAP-1 may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those described above.

In a further embodiment, an antagonist of HPRAP-3 may be administered to a subject to treat or prevent an immune disorder. Such a disorder may include, but is not limited to, those discussed above. In one aspect, an antibody which specifically binds HPRAP-3 may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HPRAP-3.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HPRAP-3 may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those described above.

In a further embodiment, an antagonist of HPRAP-4 may be administered to a subject to treat or prevent an immune disorder. Such a disorder may include, but is not limited to, those discussed above. In one aspect, an antibody which specifically binds HPRAP-4 may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HPRAP-4.

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HPRAP-4 may be administered to a subject to treat or prevent an immune disorder including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

An antagonist of HPRAP may be produced using methods which are generally known in the art. In particular, purified HPRAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HPRAP. Antibodies to HPRAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HPRAP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to HPRAP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HPRAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.

Monoclonal antibodies to HPRAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimeric antibodies,” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HPRAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HPRAP may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between HPRAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HPRAP epitopes is preferred, but a competitive binding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encoding HPRAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding HPRAP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HPRAP. Thus, complementary molecules or fragments may be used to modulate HPRAP activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HPRAP.

Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HPRAP. (See, e.g., Sambrook, supra; and Ausubel, supra.)

Genes encoding HPRAP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding HPRAP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the gene encoding HPRAP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HPRAP.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HPRAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′-O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HPRAP, antibodies to HPRAP, and mimetics, agonists, antagonists, or inhibitors of HPRAP. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of HPRAP, such labeling would include amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of active ingredient, for example HPRAP or fragments thereof, antibodies of HPRAP, and agonists, antagonists or inhibitors of HPRAP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₅₀ (the dose therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics. The dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind HPRAP may be used for the diagnosis of disorders characterized by expression of HPRAP, or in assays to monitor patients being treated with HPRAP or agonists, antagonists, or inhibitors of HPRAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HPRAP include methods which utilize the antibody and a label to detect HPRAP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.

A variety of protocols for measuring HPRAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of HPRAP expression. Normal or standard values for HPRAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HPRAP under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of HPRAP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding HPRAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HPRAP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of HPRAP, and to monitor regulation of HPRAP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HPRAP or closely related molecules may be used to identify nucleic acid sequences which encode HPRAP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding HPRAP, allelic variants, or related sequences.

Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the HPRAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequences of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 or from genomic sequences including promoters, enhancers, and introns of the HPRAP gene.

Means for producing specific hybridization probes for DNAs encoding HPRAP include the cloning of polynucleotide sequences encoding HPRAP or HPRAP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ³²p or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding HPRAP may be used for the diagnosis of a disorder associated with expression of HPRAP. Examples of such a disorder include, but are not limited to, cell proliferative disorders such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and immune disorders such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. The polynucleotide sequences encoding HPRAP may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered HPRAP expression. Such qualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HPRAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding HPRAP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HPRAP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associated with expression of HPRAP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HPRAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Additional diagnostic uses for oligonucleotides designed from the sequences encoding HPRAP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding HPRAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding HPRAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HPRAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encoding HPRAP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology and Biotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding HPRAP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, HPRAP, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HPRAP and the agent being tested may be measured.

Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with HPRAP, or fragments thereof, and washed. Bound HPRAP is then detected by methods well known in the art. Purified HPRAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HPRAP specifically compete with a test compound for binding HPRAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HPRAP.

In additional embodiments, the nucleotide sequences which encode HPRAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.

EXAMPLES I. cDNA Library Construction

THP1NOB01

The THP1NOB01 Library was constructed using RNA isolated from cultured, unstimulated THP-1 cells. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia. The THP1NOB01 cDNA library was custom constructed in the Uni-ZAP vector system by Stratagene (La Jolla, Calif.).

BLADNOT04

The BLADNOT04 Library was constructed using 1 microgram of polyA RNA isolated from bladder tissue of a 28-year-old Caucasian male who died from a self-inflicted gunshot wound (specimen #RA95-09-0677; International Institute of Advanced Medicine, Exton, Pa.). The tissue donor had a history of smoking and substance abuse.

BEPINOT01

The BEPINOT01 1 Library was constructed using 1.1 micrograms of polyA RNA isolated from a bronchial epithelium (NHBE) primary cell line derived from a 54-year-old Caucasian male.

OVARNOT03

The OVARNOT03 Library was constructed using 1 microgram of polyA RNA isolated from nontumorous ovarian tissue removed from a 43-year-old Caucasian female during a bilateral salpingo-oopherectomy (removal of the fallopian tubes and ovaries). Family history included atherosclerotic coronary artery disease, cerebrovascular disease, and breast, pancreatic and uterine cancer.

BLADNOT04, BEPINOT01, and OVARNOT03

The frozen tissue was homogenized and lysed using a POLYTRON homogenizer PT-3000 (Brinkmann Instruments, Westbury, N.J.) in guanidinium isothiocyanate solution. The lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a L8-70M ultracentrifuge (Beckman Coulter Fullerton, Calif.) for 18 hours at 25,000 rpm at ambient temperature. The RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and DNase treated at 37° C. RNA extraction and precipitation were repeated as before. The mRNA was then isolated with the OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.) and used to construct the cDNA library.

The mRNAs were handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Cat. #18248-013; Life Technologies, Gaithersburg, Md.). cDNAs were fractionated on a SEPHAROSE column CL4B (Cat. #275105-01; Amersham Pharmacia Upjohn), and those cDNAs exceeding 400 bp were ligated into either the PSPORT1 (Life Technologies) or PINCY vector (Incyte Pharmaceuticals). The plasmid was subsequently transformed into DH5α or DH12S competent cells (Life Technologies).

II. Isolation and Sequencing of cDNA Clones

For BLADNOT04 and BEPINOT01, plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Catalog #26173; QIAGEN, Inc.). For OVARNOT03, plasmid or phagemid DNA was released from cells and purified using the MINIPREP Kit (Cat. No. 77468; Advanced Genetic Technologies Corporation, Gaithersburg Md.). The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #22711, Gibco BRL Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol. Biol. 94:441f), using a MICROLAB 2200 system (Hamilton, Reno, Nev.) in combination with DNA ENGINE thermal cycler (PTC200 from MJ Research, Watertown, Mass.) and ABI PRISMS DNA sequencing systems (Perkin Elmer).

III. Similarity Searching of cDNA Clones and Their Deduced Proteins

BLADNOT04, BEPINOT01, and OVARNOT03

The nucleotide sequences and/or amino acid sequences of the Sequence Listing were used to query sequences in the GenBank, SwissProt, BLOCKS, and Pima II databases. These databases, which contain previously identified and annotated sequences, were searched for regions of similarity using BLAST (Basic Local Alignment Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST was especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Other algorithms could have been used when dealing with primary sequence patterns and secondary structure gap penalties. (See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.) The sequences disclosed in this application have lengths of at least 49 nucleotides and have no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and a database sequence. BLAST evaluated the statistical significance of any matches found, and reported only those matches that satisfy the user-selected threshold of significance. In this application, threshold was set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

Incyte nucleotide sequences were searched against the GenBank databases for primate (pri), rodent (rod), and other mammalian sequences (mam), and deduced amino acid sequences from the same clones were then searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp), for similarity.

THP1NOB01

Each cDNA was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incorporated into the INHERIT 670 sequence analysis system (Applied Biosystems). In this algorithm, Pattern Specification Language (TRW Inc, Los Angeles Calif.) was used to determine regions of homology. The three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance. Using a combination of these three parameters, the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-Waterman alignments were used to display the results of the homology search.

Peptide and protein sequence homologies were ascertained using the INHERIT-670 sequence analysis system in a way similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches.

Additionally, sequences identified from cDNA libraries may be analyzed to identify those gene sequences encoding conserved protein motifs using an appropriate analysis program, e.g., BLOCKS. BLOCKS is a weighted matrix analysis algorithm based on short amino acid segments, or blocks, compiled from the PROSITE database. (Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221.) The BLOCKS algorithm is useful for classifying genes with unknown functions. (Henikoff S. and Henikoff G. P. (1991) Nucleic Acids Res. 19:6565-6572.) Blocks, which are 3-60 amino acids in length, correspond to the most highly conserved regions of proteins. The BLOCKS algorithm compares a query sequence with a weighted scoring matrix of blocks in the BLOCKS database. Blocks in the BLOCKS database are calibrated against protein sequences with known functions from the SWISS-PROT database to determine the stochastic distribution of matches. Similar databases such as PRINTS, a protein fingerprint database, are also searchable using the BLOCKS algorithm. (Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37:417-424.) PRINTS is based on non-redundant sequences obtained from sources such as SWISS-PROT, GenBank, PIR, and NRL-3D.

The BLOCKS algorithm searches for matches between a query sequence and the BLOCKS or PRINTS database and evaluates the statistical significance of any matches found. Matches from a BLOCKS or PRINTS search can be evaluated on two levels, local similarity and global similarity. The degree of local similarity is measured by scores, and the extent of global similarity is measured by score ranking and probability values. A score of 1000 or greater for a BLOCKS match of highest ranking indicates that the match falls within the 0.5 percentile level of false positives when the matched block is calibrated against SWISS-PROT. Likewise, a probability value of less than 1.0×10⁻³ indicates that the match would occur by chance no more than one time in every 1000 searches. Only those matches with a cutoff score of 1000 or greater and a cutoff probability value of 1.0×10⁻³ or less are considered in the functional analyses of the protein sequences in the Sequence Listing.

Nucleic and amino acid sequences of the Sequence Listing may also be analyzed using PFAM. PFAM is a Hidden Markov Model (HMM) based protocol useful in protein family searching. HMM is a probalistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.)

The PFAM database contains protein sequences of 527 protein families gathered from publicly available sources, e.g., SWISS-PROT and PROSITE. PFAM searches for well characterized protein domain families using two high-quality alignment routines, seed alignment and full alignment. (See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins 28:405-420.) The seed alignment utilizes the hmmls program, a program that searches for local matches, and a non-redundant set of the PFAM database. The full alignment utilizes the hmmfs program, a program that searches for multiple fragments in long sequences, e.g., repeats and motifs, and all sequences in the PFAM database. A result or score of 100 “bits” can signify that it is 2¹⁰⁰-fold more likely that the sequence is a true match to the model or comparison sequence. Cutoff scores which range from 10 to 50 bits are generally used for individual protein families using the SWISS-PROT sequences as model or comparison sequences.

Two other algorithms, SIGPEPT and TM, both based on the HMM algorithm described above (see, e.g., Eddy, supra; and Sonnhammer, supra), identify potential signal sequences and transmembrane domains, respectively. SIGPEPT was created using protein sequences having signal sequence annotations derived from SWISS-PROT. It contains about 1413 non-redundant signal sequences ranging in length from 14 to 36 amino acid residues. TM was created similarly using transmembrane domain annotations. It contains about 453 non-redundant transmembrane sequences encompassing 1579 transmembrane domain segments. Suitable HMM models were constructed using the above sequences and were refined with known SWISS-PROT signal peptide sequences or transmembrane domain sequences until a high correlation coefficient, a measurement of the correctness of the analysis, was obtained. Using the protein sequences from the SWISS-PROT database as a test set, a cutoff score of 11 bits, as determined above, correlated with 91-94% true-positives and about 4.1% false-positives, yielding a correlation coefficient of about 0.87-0.90 for SIGPEPT. A score of 11 bits for TM will typically give the following results: 75% true positives; 1.72% false positives; and a correlation coefficient of 0.76. Each search evaluates the statistical significance of any matches found and reports only those matches that score at least 11 bits.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.

The basis of the search is the product score, which is defined as: $\frac{{\% \quad {sequence}\quad {identity}\quad \times \quad \% \quad {maximum}\quad {BLAST}\quad {score}}\quad}{100}$

The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.

The results of Northern analysis are reported as a list of libraries in which the transcript encoding HPRAP occurs. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library.

V. Extension of HPRAP Encoding Polynucleotides

The nucleic acid sequences of Incyte Clones 031381, 1319265, 2057812, and 2058485 were used to design oligonucleotide primers for extending partial nucleotide sequences to full length. For each nucleic acid sequence, one primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide. Primers were used to facilitate the extension of the known sequence “outward” generating amplicons containing new unknown nucleotide sequence for the region of interest. The initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences, Plymouth, Minn.), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.

Selected human cDNA libraries (GIBCO BRL) were used to extend the sequence. If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was performed using the DNA ENGINE thermal cycler (PTC200; M.J. Research, Watertown, Mass.), beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6% to 0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQUICK (QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16° C. Competent E. coli cells (in 40 μl of appropriate media) were transformed with 3 μl of ligation mixture and cultured in 80 μl of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37° C., the E. coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2× carb). The following day, several colonies were randomly picked from each plate and cultured in 150 μl of liquid LB/2× carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 μl of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (and holding)

Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and appropriate clones were selected, ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 are used to obtain 5′ regulatory sequences using the procedure above, oligonucleotides designed for 5′ extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosine triphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquot containing 10⁷ counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film (Eastman Kodak) is exposed to the blots for several hours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; and Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the HPRAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HPRAP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software and the coding sequence of HPRAP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HPRAP-encoding transcript.

IX. Expression of HPRAP

Expression and purification of HPRAP is achieved using bacterial or virus-based expression systems. For expression of HPRAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express HPRAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of HPRAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica califomica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HPRAP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)

In most expression systems, HPRAP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Pharmacia, Piscataway, N.J.). Following purification, the GST moiety can be proteolytically cleaved from HPRAP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN Inc, Chatsworth, Calif.). Methods for protein expression and purification are discussed in Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10, 16. Purified HPRAP obtained by these methods can be used directly in the following activity assay.

X. Demonstration of HPRAP Activity

The protease activity of HPRAP-1 and HPRAP-4 is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantitated by spectrophotometric (or fluorometric) absorption of the released chromophore. (Beynon, R. J. and J. S. Bond (1994) Proteolytic Enzymes: A Practical Approach. Oxford University Press, New York, N.Y., pp.25-55.) Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases), aminopeptidase (leucine aminopeptidase), or carboxypeptidase (Carboxypeptidase A and B, procollagen C-proteinase). Chromogens commonly used are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid. Assays are performed at-ambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette and followed by the increase/decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate. The change in absorbance is proportional to the enzyme activity in the assay.

The assay for HPRAP-3 is carried out as described above using as the source of protease activity, a proteasome complex reconstituted with HPRAP-3 in the absence of the p27 subunit.

The assay for HPRAP-2 is carried out as described above for HPRAP-1 and HPRAP-4 using a serine protease assayed in the absence and in the presence of various concentrations of HPRAP-2. Inhibition of serine protease activity is proportional to the activity of HPRAP-2 in the assay.

XI. Functional Assays

HPRAP function is assessed by expressing the sequences encoding HPRAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT vector (Life Technologies, Gaithersburg, Md.) and PCR 3.1 vector (Invitrogen, Carlsbad, Calif.), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York, N.Y.

The influence of HPRAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HPRAP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding HPRAP and other genes of interest can be analyzed by Northern analysis or microarray techniques.

XII. Production of HPRAP Specific Antibodies

HPRAP substantially purified using polyacrylamide gel electrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.

Alternatively, the HPRAP amino acid sequence is analyzed using LASERGENE software (DNASTAR Inc.) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Perkin Elmer) using fmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

XIII. Purification of Naturally Occurring HPRAP Using Specific Antibodies

Naturally occurring or recombinant HPRAP is substantially purified by immunoaffinity chromatography using antibodies specific for HPRAP. An immunoaffinity column is constructed by covalently coupling anti-HPRAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing HPRAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HPRAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/HPRAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HPRAP is collected.

XIV. Identification of Molecules Which Interact with HPRAP

HPRAP, or biologically active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HPRAP, washed, and any wells with labeled HPRAP complex are assayed. Data obtained using different concentrations of HPRAP are used to calculate values for the number, affinity, and association of HPRAP with the candidate molecules.

Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

12 459 amino acids amino acid single linear unknown THP1NOB01 031381 1 Met Asp Gly Glu Asp Ile Pro Asp Phe Ser Ser Leu Lys Glu Glu 5 10 15 Thr Ala Tyr Trp Lys Glu Leu Ser Leu Lys Tyr Lys Gln Ser Phe 20 25 30 Gln Glu Ala Arg Asp Glu Leu Val Glu Phe Gln Glu Gly Ser Arg 35 40 45 Glu Leu Glu Ala Glu Leu Glu Ala Gln Leu Val Gln Ala Glu Gln 50 55 60 Arg Asn Arg Asp Leu Gln Ala Asp Asn Gln Arg Leu Lys Tyr Glu 65 70 75 Val Glu Ala Leu Lys Glu Lys Leu Glu His Gln Tyr Ala Gln Ser 80 85 90 Tyr Lys Gln Val Ser Val Leu Glu Asp Asp Leu Ser Gln Thr Arg 95 100 105 Ala Ile Lys Glu Gln Leu His Lys Tyr Val Arg Glu Leu Glu Gln 110 115 120 Ala Asn Asp Asp Leu Glu Arg Ala Lys Arg Ala Thr Ile Val Ser 125 130 135 Leu Glu Asp Phe Glu Gln Arg Leu Asn Gln Ala Ile Glu Arg Asn 140 145 150 Ala Phe Leu Glu Ser Glu Leu Asp Glu Lys Glu Ser Leu Leu Val 155 160 165 Ser Val Gln Arg Leu Lys Asp Glu Ala Arg Asp Leu Arg Gln Glu 170 175 180 Leu Ala Val Arg Glu Arg Gln Gln Glu Val Thr Arg Lys Ser Ala 185 190 195 Pro Ser Ser Pro Thr Leu Asp Cys Glu Lys Met Asp Ser Ala Val 200 205 210 Gln Ala Ser Leu Ser Leu Pro Ala Thr Pro Val Gly Lys Gly Thr 215 220 225 Glu Asn Thr Phe Pro Ser Pro Lys Ala Ile Pro Asn Gly Phe Gly 230 235 240 Thr Ser Pro Leu Thr Pro Ser Ala Arg Ile Ser Ala Leu Asn Ile 245 250 255 Val Gly Asp Leu Leu Arg Lys Val Gly Ala Leu Glu Ser Lys Leu 260 265 270 Ala Ala Cys Arg Asn Phe Ala Lys Asp Gln Ala Ser Arg Lys Ser 275 280 285 Tyr Ile Ser Gly Asn Val Asn Cys Gly Val Leu Asn Gly Asn Gly 290 295 300 Thr Lys Phe Ser Arg Ser Gly His Thr Ser Phe Phe Asp Lys Gly 305 310 315 Ala Val Asn Gly Phe Asp Pro Ala Pro Pro Pro Pro Gly Leu Gly 320 325 330 Ser Ser Arg Pro Ser Ser Ala Pro Gly Met Leu Pro Leu Ser Val 335 340 345 Arg Val Pro Ser Leu Gln Val Gly Ala Pro Ala Leu Leu Gln Gln 350 355 360 Pro Arg Thr Pro Thr Pro His Pro Ser Val Pro Gly Pro Ser Pro 365 370 375 Val Pro Leu Arg Leu Pro Pro His Gly Trp Gln Arg Ala Gly Cys 380 385 390 Met Gln Trp Arg Leu Leu Gly Pro Ala Gln Pro Arg Asn Ser Ala 395 400 405 Arg Tyr Gln Tyr Trp Leu Phe Ser Leu Leu Ala Val Val Pro Leu 410 415 420 Val Ser His Asp Cys Thr Phe Val Gly Arg Lys Val Ile His Thr 425 430 435 Cys Ile Thr Trp Ser Leu Asp Ala Glu Val Pro Ile His His Thr 440 445 450 Cys Pro Ile Ala Pro Thr Leu Leu Tyr 455 348 amino acids amino acid single linear unknown BLADNOT04 1319265 2 Met Trp Lys Thr Gln Ile Gly Ala Tyr Cys Gly Val Thr Thr Asp 5 10 15 Val Arg Val Glu Arg Lys Asp Pro Asn Gln Val Glu Leu Trp Gly 20 25 30 Leu Lys Glu Gly Thr Tyr Leu Phe Gln Leu Thr Val Thr Ser Ser 35 40 45 Asp His Pro Glu Asp Thr Ala Asn Val Thr Val Thr Val Leu Ser 50 55 60 Thr Lys Gln Thr Glu Asp Tyr Cys Leu Ala Ser Asn Lys Val Gly 65 70 75 Arg Arg Cys Arg Gly Ser Phe Pro Arg Trp Tyr Tyr Asp Pro Thr 80 85 90 Glu Gln Ile Cys Lys Ser Phe Val Tyr Gly Gly Cys Leu Gly Asn 95 100 105 Lys Asn Asn Tyr Leu Arg Glu Glu Glu Cys Ile Leu Ala Cys Arg 110 115 120 Gly Val Gln Gly Gly Pro Leu Arg Gly Ser Ser Gly Ala Gln Ala 125 130 135 Thr Phe Pro Gln Gly Pro Ser Met Glu Arg Arg His Pro Val Cys 140 145 150 Ser Gly Thr Cys Gln Pro Thr Gln Phe Arg Cys Ser Asn Gly Cys 155 160 165 Cys Ile Asp Ser Phe Leu Glu Cys Asp Asp Thr Pro Asn Cys Pro 170 175 180 Asp Ala Ser Asp Glu Ala Ala Cys Glu Lys Tyr Thr Ser Gly Phe 185 190 195 Asp Glu Leu Gln Arg Ile His Phe Pro Ser Asp Lys Gly His Cys 200 205 210 Val Asp Leu Pro Asp Thr Gly Leu Cys Lys Glu Ser Ile Pro Arg 215 220 225 Trp Tyr Tyr Asn Pro Phe Ser Glu His Cys Ala Arg Phe Thr Tyr 230 235 240 Gly Gly Cys Tyr Gly Asn Lys Asn Asn Phe Glu Glu Glu Gln Gln 245 250 255 Cys Leu Glu Ser Cys Arg Gly Ile Ser Lys Lys Asp Val Phe Gly 260 265 270 Leu Arg Arg Glu Ile Pro Ile Pro Ser Thr Gly Ser Val Glu Met 275 280 285 Ala Val Ala Val Phe Leu Val Ile Cys Ile Val Val Val Val Ala 290 295 300 Ile Leu Gly Tyr Cys Phe Phe Lys Asn Gln Arg Lys Asp Phe His 305 310 315 Gly His His His His Pro Pro Pro Thr Pro Ala Ser Ser Thr Val 320 325 330 Ser Thr Thr Glu Asp Thr Glu His Leu Val Tyr Asn His Thr Thr 335 340 345 Arg Pro Leu 238 amino acids amino acid single linear unknown BEPINOT01 2057812 3 Met Ser Asp Glu Glu Ala Arg Gln Ser Gly Gly Ser Ser Gln Ala 5 10 15 Gly Ala Val Thr Val Ser Asp Val Gln Glu Leu Met Arg Arg Lys 20 25 30 Glu Glu Ile Glu Ala Gln Ile Lys Ala Asn Tyr Asp Val Leu Glu 35 40 45 Ser Gln Lys Gly Ile Gly Met Asn Glu Pro Leu Val Asp Cys Glu 50 55 60 Gly Tyr Pro Arg Ser Asp Val Asp Leu Tyr Gln Val Arg Thr Ala 65 70 75 Arg His Asn Ile Ile Cys Leu Gln Asn Asp His Lys Ala Val Met 80 85 90 Lys Gln Val Glu Glu Ala Leu His Gln Leu His Ala Arg Asp Lys 95 100 105 Glu Lys Gln Ala Arg Asp Met Ala Glu Ala His Lys Glu Ala Met 110 115 120 Ser Arg Lys Leu Gly Gln Ser Glu Ser Gln Gly Pro Pro Arg Ala 125 130 135 Phe Ala Lys Val Asn Ser Ile Ser Pro Gly Ser Pro Ala Ser Ile 140 145 150 Ala Gly Asn Pro Gly Val Gly His Ser Ser Pro Cys Pro Gly Asp 155 160 165 Thr Gly Leu Gln Val Asp Asp Glu Ile Val Glu Phe Gly Ser Val 170 175 180 Asn Thr Gln Asn Phe Gln Ser Leu His Asn Ile Gly Ser Val Val 185 190 195 Gln His Ser Glu Gly Lys Pro Leu Asn Val Thr Val Ile Arg Arg 200 205 210 Gly Gly Lys His Gln Leu Arg Leu Val Pro Thr Arg Trp Ala Gly 215 220 225 Lys Gly Leu Leu Gly Cys Asn Ile Ile Pro Leu Gln Arg 230 235 471 amino acids amino acid single linear unknown OVARNOT03 2058485 4 Met Asn Gly Lys Ala Arg Lys Glu Ala Val Gln Thr Ala Ala Lys 5 10 15 Glu Leu Leu Lys Phe Val Asn Arg Ser Pro Ser Pro Phe His Ala 20 25 30 Val Ala Glu Cys Arg Asn Arg Leu Leu Gln Ala Gly Phe Ser Glu 35 40 45 Leu Lys Glu Thr Glu Lys Trp Asn Ile Lys Pro Glu Ser Lys Tyr 50 55 60 Phe Met Thr Arg Asn Ser Ser Thr Ile Ile Ala Phe Ala Val Gly 65 70 75 Gly Gln Tyr Val Pro Gly Asn Gly Phe Ser Leu Ile Gly Ala His 80 85 90 Thr Asp Ser Pro Cys Leu Arg Val Lys Arg Arg Ser Arg Arg Ser 95 100 105 Gln Val Gly Phe Gln Gln Val Gly Val Glu Thr Tyr Gly Gly Gly 110 115 120 Ile Trp Ser Thr Trp Phe Asp Arg Asp Leu Thr Leu Ala Gly Arg 125 130 135 Val Ile Val Lys Cys Pro Thr Ser Gly Arg Leu Glu Gln Gln Leu 140 145 150 Val His Val Glu Arg Pro Ile Leu Arg Ile Pro His Leu Ala Ile 155 160 165 His Leu Gln Arg Asn Ile Asn Glu Asn Phe Gly Pro Asn Thr Glu 170 175 180 Met His Leu Val Pro Ile Leu Ala Thr Ala Ile Gln Glu Glu Leu 185 190 195 Glu Lys Gly Thr Pro Glu Pro Gly Pro Leu Asn Ala Val Asp Glu 200 205 210 Arg His His Ser Val Leu Met Ser Leu Leu Cys Ala His Leu Gly 215 220 225 Leu Ser Pro Lys Asp Ile Val Glu Met Glu Leu Cys Leu Ala Asp 230 235 240 Thr Gln Pro Ala Val Leu Gly Gly Ala Tyr Asp Glu Phe Ile Phe 245 250 255 Ala Pro Arg Leu Asp Asn Leu His Ser Cys Phe Cys Ala Leu Gln 260 265 270 Ala Leu Ile Asp Ser Cys Ala Gly Pro Gly Ser Leu Ala Thr Glu 275 280 285 Pro His Val Arg Met Val Thr Leu Tyr Asp Asn Glu Glu Val Gly 290 295 300 Ser Glu Ser Ala Gln Gly Ala Gln Ser Leu Leu Thr Glu Leu Val 305 310 315 Leu Arg Arg Ile Ser Ala Ser Cys Gln His Pro Thr Ala Phe Glu 320 325 330 Glu Ala Ile Pro Lys Ser Phe Met Ile Ser Ala Asp Met Ala His 335 340 345 Ala Val His Pro Asn Tyr Leu Asp Lys His Glu Glu Asn His Arg 350 355 360 Pro Leu Phe His Lys Gly Pro Val Ile Lys Val Asn Ser Lys Gln 365 370 375 Arg Tyr Ala Ser Asn Ala Val Ser Glu Ala Leu Ile Arg Glu Val 380 385 390 Ala Asn Lys Val Lys Val Pro Leu Gln Asp Leu Met Val Arg Asn 395 400 405 Asp Thr Pro Cys Gly Thr Thr Ile Gly Pro Ile Leu Ala Ser Arg 410 415 420 Leu Gly Leu Arg Val Leu Asp Leu Gly Ser Pro Gln Leu Ala Met 425 430 435 His Ser Ile Arg Glu Met Ala Cys Thr Thr Gly Val Leu Gln Thr 440 445 450 Leu Thr Leu Phe Lys Gly Phe Phe Glu Leu Phe Pro Ser Leu Ser 455 460 465 His Asn Leu Leu Val Asp 470 2327 base pairs nucleic acid single linear unknown THP1NOB01 031381 5 CGGGCGCGGA GGTACGCTGA GTGGAGCTCG GGGCTGCGTA GGGGAGCTGA GCCGAGCGGC 60 TGGGCGGGCC TGGCCGGGCC AGCGGAGGGG AGACGTCGGT TGAGCGGCGG CGAACATGCG 120 CTTTTGACAC ATTGGAGGCT TTCTTGATCA TGGATGGTGA AGATATACCA GATTTTTCAA 180 GTTTAAAGGA GGAAACTGCT TATTGGAAGG AACTTTCCTT GAAGTATAAG CAAAGCTTCC 240 AGGAAGCTCG GGATGAGCTA GTTGAATTCC AGGAAGGAAG CAGAGAATTA GAAGCAGAGT 300 TGGAGGCACA ATTAGTACAG GCTGAACAAA GAAATAGAGA CTTGCAGGCT GATAACCAAA 360 GACTGAAATA TGAAGTGGAG GCATTAAAGG AGAAGCTAGA GCATCAATAT GCACAGAGCT 420 ATAAGCAGGT CTCAGTGTTA GAAGATGATT TAAGTCAGAC TCGGGCCATT AAGGAGCAGT 480 TGCATAAGTA TGTGAGAGAG CTGGAGCAGG CCAACGACGA CCTGGAGCGA GCCAAAAGGG 540 CAACAATAGT TTCACTGGAA GACTTTGAAC AAAGGCTAAA CCAGGCCATT GAACGAAATG 600 CATTTTTAGA AAGTGAACTT GATGAAAAGG AATCTTTGTT GGTCTCTGTA CAGAGGTTAA 660 AGGATGAAGC AAGAGATTTA AGGCAAGAAC TAGCAGTTCG GGAAAGACAA CAGGAAGTAA 720 CTAGAAAGTC GGCTCCTAGC TCTCCAACTC TAGACTGTGA AAAGATGGAC TCCGCCGTCC 780 AAGCATCACT TTCTTTGCCA GCTACCCCTG TTGGCAAAGG AACGGAGAAC ACTTTTCCTT 840 CACCGAAAGC TATACCAAAT GGTTTTGGTA CCAGTCCACT AACTCCCTCT GCTAGGATAT 900 CAGCACTAAA CATCGTGGGG GATCTCTTAC GGAAAGTAGG GGCTTTAGAA TCCAAATTAG 960 CAGCTTGCAG GAATTTTGCA AAGGACCAAG CATCACGAAA ATCCTATATT TCAGGGAATG 1020 TTAACTGTGG GGTGCTGAAT GGCAATGGCA CAAAGTTCTC TCGATCAGGG CATACATCTT 1080 TCTTCGACAA AGGGGCAGTA AACGGCTTTG ACCCCGCTCC TCCTCCTCCT GGTCTGGGCT 1140 CCTCGCGTCC ATCGTCAGCG CCGGGTATGC TGCCTCTCAG TGTGCGAGTG CCTAGCCTCC 1200 AGGTGGGGGC TCCTGCCCTC CTCCAACAAC CCAGGACACC CACGCCTCAC CCCTCGGTGC 1260 CTGGGCCCAG CCCCGTGCCC CTCCGTCTGC CTCCGCACGG CTGGCAGAGG GCAGGCTGCA 1320 TGCAGTGGCG GCTACTGGGC CCTGCCCAGC CCCGGAACTC TGCGCGATAT CAATACTGGC 1380 TATTTTCTCT TCTCGCCGTA GTGCCGTTGG TTTCACATGA TTGCACTTTT GTGGGTCGCA 1440 AGGTGATACA TACGTGTATT ACTTGGTCAC TGGATGCAGA AGTACCCATT CATCACACCT 1500 GCCCCATAGC CCCCACTCTG CTGTACTGAT AGGATTTAGT TGTGTTTTAG GACATTGCAA 1560 ATCTTCTAGA AGTTCTCCCC CAAATCAGGT CAATGTGTGC CCTCCTGAGC TCCCACCCAG 1620 GCATCTCCAG TGCTCATGAT CATGTGTCCC CCAACTCCAC CCCTCACAGT TTGGGCCTGT 1680 TTCTGGCAAA GAGTCAGGAA GGTTACTGAA TTAGGGAACA TTTTCTGCAC CTTCTGATTT 1740 TACTTAAGCA GCTACCATTC CATGGACTTG CCTCCCAGAG CAGCACAATG CCCGTCTGAG 1800 CCCCACGTGG CAGGAGCCTC TGGGACGGGG CACACACAGG CCCAGCCTCT GTGCTGTCTC 1860 CTCCTCTGTG CGCCTCAGAC TCGGGGTGAG GGAGGCGGGC AGCCTCTCGC CAGCCTTCCC 1920 GTCCTTCAGT TCAACGACAT CTTTGGAGTG TTTTTGTTTT CTCTTCCAAG GGCCGTCCCG 1980 TTGTGTTAGG AAGGGTGAGT GGCTGGTTCC AGGGTGGGCC GGTGCCAGCT CCGGGGTGGA 2040 CTGAACAGCG GCGGCTGTCC CTGTGCATCC TTTGATTACT CTCATGCTGC ATTTACTGTT 2100 TACATTTGTT TTATTGTACA TAGGTTTGTA AACATTATTG CCTGAGATAT TTGTATATAA 2160 CTTGGGCTTT GTAGCTTTTA TTTATTCAGA ACGCATACGG CATGTTAATG ACTCTGATGG 2220 TGTCCTCCTC TGGGCAGCTG TATAGGATCA TCATGTGGTT ACAAAAAATA CTTCCCTCAA 2280 AAAAATTCTT TTAATGTGGA AACAATAAAT TTCACAGAAA AAAAAAA 2327 1870 base pairs nucleic acid single linear unknown BLADNOT04 1319265 6 CTGAGAGAAG CCTGGTCCAT CTAGTGAGAA TTGACCTTAT CTCACTTTCT CTCCCCGCCA 60 GGGTCTGGGA TCCCCAAGGC CTGGGCAGGC ATAGACTTGA AGGTACAACC CCAGGAACCC 120 CTGGTGCTGA AGGATGTGGA AAACACAGAT TGGCGCCTAC TGCGGGGTGA CAACGGATGT 180 CAGGGTAGAG AGGAAAGACC CAAACCAGGT GGAACTGTGG GGACTCAAGG AAGGCACCTA 240 CCTGTTCCAG CTGACAGTGA CTAGCTCAGA CCACCCAGAG GACACGGCCA ACGTCACAGT 300 CACTGTGCTG TCCACCAAGC AGACAGAAGA CTACTGCCTC GCATCCAACA AGGTGGGTCG 360 CCGCTGCCGG GGCTCTTTCC CGCGCTGGTA CTATGACCCC ACGGAGCAGA TCTGCAAGAG 420 TTTCGTTTAT GGAGGCTGCT TGGGCAACAA GAACAACTAC CTTCGGGAAG AAGAGTGCAT 480 TCTAGCCTGT CGGGGTGTGC AAGGTGGGCC TTTGAGAGGC AGCTCTGGGG CTCAGGCGAC 540 TTTCCCCCAG GGCCCCTCCA TGGAAAGGCG CCATCCAGTG TGCTCTGGCA CCTGTCAGCC 600 CACCCAGTTC CGCTGCAGCA ATGGCTGCTG CATCGACAGT TTCCTGGAGT GTGACGACAC 660 CCCCAACTGC CCCGACGCCT CCGACGAGGC TGCCTGTGAA AAATACACGA GTGGCTTTGA 720 CGAGCTCCAG CGCATCCATT TCCCCAGTGA CAAAGGGCAC TGCGTGGACC TGCCAGACAC 780 AGGACTCTGC AAGGAGAGCA TCCCGCGCTG GTACTACAAC CCCTTCAGCG AACACTGCGC 840 CCGCTTTACC TATGGTGGTT GTTATGGCAA CAAGAACAAC TTTGAGGAAG AGCAGCAGTG 900 CCTCGAGTCT TGTCGCGGCA TCTCCAAGAA GGATGTGTTT GGCCTGAGGC GGGAAATCCC 960 CATTCCCAGC ACAGGCTCTG TGGAGATGGC TGTCGCAGTG TTCCTGGTCA TCTGCATTGT 1020 GGTGGTGGTA GCCATCTTGG GTTACTGCTT CTTCAAGAAC CAGAGAAAGG ACTTCCACGG 1080 ACACCACCAC CACCCACCAC CCACCCCTGC CAGCTCCACT GTCTCCACTA CCGAGGACAC 1140 GGAGCACCTG GTCTATAACC ACACCACCCG GCCCCTCTGA GCCTGGGTCT CACCGGCTCT 1200 CACCTGGCCC TGCTTCCTGC TTGCCAAGGC AGAGGCCTGG GCTGGGAAAA ACTTTGGAAC 1260 CAGACTCTTG CCTGTTTCCC AGGCCCACTG TGCCTCAGAG ACCAGGGCTC CAGCCCCTCT 1320 TGGAGAAGTC TCAGCTAAGC TCACGTCCTG AGAAAGCTCA AAGGTTTGGA AGGAGCAGAA 1380 AACCCTTGGG CCAGAAGTAC CAGACTAGAT GGACCTGCCT GCATAGGAGT TTGGAGGAAG 1440 TTGGAGTTTT GTTTCCTCTG TTCAAAGCTG CCTGTCCCTA CCCCATGGTG CTAGGAAGAG 1500 GAGTGGGGTG GTGTCAGACC CTGGAGGCCC CAACCCTGTC CTCCCGAGCT CCTCTTCCAT 1560 GCTGTGCGCC CAGGGCTGGG AGGAAGGACT TCCCTGTGTA GTTTGTGCTG TAAAGAGTTG 1620 CTTTTTGTTT ATTTAATGCT GTGGCATGGG TGAAGAGGAG GGGAAGAGGC CTGTTTGGCC 1680 TCTCTGTCCT CTCTTCCTCT TCCCCCAAGA TTGAGCTCTC TGCCCTTGAT CAGCCCCACC 1740 CTGGCCTAGA CCAGCAGACA GAGCCAGGAG AGGCTCAGCT GCATTCCGCA GCCCCCACCC 1800 CCAAGGTTCT CCAACATCAC AGCCCAGCCC ACCCACTGGG TAATAAAAGT GGTTTGTGGA 1860 AAAAAAAAAA 1870 1138 base pairs nucleic acid single linear unknown BEPINOT01 2057812 7 GTTTTGGCGC ATGGGCGGAG CGTAGTTACG GTCGACTGGG GCGTCGTCCC TAGCCCGGGA 60 GCCGGGTCTC TGGAGTCGCG GCCCGGGGTT CACGATGTCC GACGAGGAAG CGAGGCAGAG 120 CGGAGGCTCC TCGCAGGCCG GCGCCGTGAC TGTCAGCGAC GTCCAGGAGC TGATGCGGCG 180 CAAGGAGGAG ATAGAAGCGC AGATCAAGGC CAACTATGAC GTGCTGGAAA GCCAAAAAGG 240 CATTGGGATG AACGAGCCGC TGGTGGACTG TGAGGGCTAC CCCCGGTCAG ACGTGGACCT 300 GTACCAAGTC CGCACCGCCA GGCACAACAT CATATGCCTG CAGAATGATC ACAAGGCAGT 360 GATGAAGCAG GTGGAGGAGG CCCTGCACCA GCTGCACGCT CGCGACAAGG AGAAGCAGGC 420 CCGGGACATG GCTGAGGCCC ACAAAGAGGC CATGAGCCGC AAACTGGGTC AGAGTGAGAG 480 CCAGGGCCCT CCACGGGCCT TCGCCAAAGT GAACAGCATC AGCCCCGGCT CCCCAGCCAG 540 CATCGCGGGT AATCCAGGGG TTGGCCACTC AAGTCCATGC CCAGGGGACA CGGGTCTGCA 600 AGTGGATGAT GAGATTGTGG AGTTCGGCTC TGTGAACACC CAGAACTTCC AGTCACTGCA 660 TAACATTGGC AGTGTGGTGC AGCACAGTGA GGGGAAGCCC CTGAATGTGA CAGTGATCCG 720 CAGGGGGGGA AAACACCAGC TTAGACTTGT TCCAACACGC TGGGCAGGAA AAGGACTGCT 780 GGGCTGCAAC ATTATTCCTC TGCAAAGATG ATTGTCCCTG GGGAACAGTA ACAGGAAAGC 840 ATCTTCCCTT GCCCTGGACT TGGGTCTAGG GATTTCCAAC TTGTCTTCTC TCCCTGAAGC 900 ATAAGGATCT GGAAGAGGCT TGTAACCTGA ACTTCTGTGT GGTGGCAGTA CTGTGGCCCA 960 CCAGTGTAAT CTCCCTGGAT TAAGGCATTC TTAAAAACTT AGGCTTGGCC TCTTTCACAA 1020 ATTAGGCCAC GGCCCTAAAT AGGAATTCCC TGGATTGTGG GCAAGTGGGC GGAAGTTATT 1080 CTGGCAGGTA CTGGTGTGAT TATTATTATT ATTTTTAATA AAGAGTTTTA CAGTGCTG 1138 1910 base pairs nucleic acid single linear unknown OVARNOT03 2058485 8 CGAGCCCGGA GGCCAGATGA GCGGACACAG CCCCACGCGC GGGGCCATGC AGGTAAGTGG 60 CTCCCGACGG CCCCACTTGA ATTTCGATCC CAGACCGGGT CCGGCGCCCT CCGGGGCCCA 120 AGCTTAGCGC GGTGCTGCAG TGGGGCCGCC TGACCCAAAG CGAAACCGAA AGCCCCGCGG 180 AGGGTGACCT GACGACTTTC CCGGGACTGG AAGGGGGAGT CCTGCGAGAG ACTAGGTGGC 240 CATGAACGGT AAGGCCCGCA AAGAGGCGGT GCAGACTGCG GCTAAGGAAC TCCTCAAGTT 300 CGTGAACCGG AGTCCCTCTC CTTTCCATGC TGTGGCTGAA TGCCGCAACC GCCTTCTCCA 360 GGCTGGCTTC AGTGAACTCA AGGAGACTGA GAAATGGAAT ATTAAGCCCG AGAGCAAGTA 420 CTTCATGACC AGGAACTCCT CCACCATCAT AGCTTTTGCT GTAGGGGGCC AGTACGTTCC 480 TGGCAATGGC TTCAGCCTCA TCGGGGCCCA CACGGACAGC CCCTGCCTCC GGGTGAAACG 540 TCGGTCTCGC CGCAGCCAGG TGGGCTTCCA GCAAGTCGGT GTGGAGACCT ATGGTGGTGG 600 GATCTGGAGC ACCTGGTTTG ACCGTGACCT GACTCTGGCT GGACGCGTCA TTGTCAAGTG 660 CCCTACCTCA GGTCGGCTGG AGCAGCAGCT GGTGCACGTG GAGCGGCCCA TTCTTCGCAT 720 CCCACACCTG GCCATCCATC TGCAGCGAAA TATCAACGAG AACTTTGGGC CCAACACAGA 780 GATGCATCTA GTCCCCATTC TTGCCACAGC CATCCAGGAG GAGCTGGAGA AGGGGACTCC 840 TGAGCCAGGG CCTCTCAATG CTGTGGATGA GCGGCACCAT TCGGTCCTCA TGTCCCTGCT 900 CTGTGCCCAT CTGGGGCTGA GCCCCAAGGA CATAGTGGAG ATGGAGCTCT GCCTTGCAGA 960 CACCCAGCCT GCGGTCTTGG GTGGTGCCTA TGATGAGTTC ATCTTTGCTC CTCGGCTGGA 1020 CAATCTGCAC AGCTGCTTCT GTGCCCTGCA GGCCTTGATA GATTCCTGTG CAGGCCCTGG 1080 CTCCCTGGCC ACAGAGCCTC ACGTGCGCAT GGTCACACTC TATGACAACG AAGAGGTGGG 1140 GTCTGAGAGT GCACAGGGAG CACAGTCACT GCTGACAGAG CTGGTGCTGC GGCGGATCTC 1200 AGCCTCGTGC CAGCACCCGA CAGCCTTCGA GGAAGCCATA CCCAAGTCCT TCATGATCAG 1260 CGCAGACATG GCCCATGCTG TGCATCCCAA CTACCTGGAC AAGCATGAGG AGAACCACCG 1320 GCCTTTATTC CACAAGGGCC CCGTGATCAA GGTGAACAGC AAGCAACGCT ATGCTTCAAA 1380 CGCGGTGTCA GAGGCCCTGA TCCGAGAGGT GGCCAACAAA GTCAAGGTCC CCCTGCAGGA 1440 TCTCATGGTC CGGAATGACA CCCCCTGTGG AACCACCATT GGACCTATCT TGGCTTCTCG 1500 GCTGGGGCTG CGGGTGCTGG ATTTAGGCAG CCCCCAACTG GCCATGCACT CTATCCGGGA 1560 GATGGCCTGC ACCACAGGAG TCCTCCAGAC CCTCACCCTC TTCAAGGGCT TCTTTGAGCT 1620 GTTCCCTTCT CTAAGCCATA ATCTCTTAGT GGATTGAGCC CTCTTGGAAA GACTTCTCTG 1680 CCATCCCTTT GCACCTGAGA GGGGAAGTTC TCAGCTGAGC TGAAGCTGGA TTATTAAAGT 1740 GGATTGTCAC TCAGACTCTC CGTGCTACGC TTATTTGGAG ACTAGAGGAG TGGGAGTTGA 1800 GCCTGGCTTG AACCTTTGGA ACCAGAAAAG TTGGGGAGCA GGTGGAGGAG GCCACACTCC 1860 TGGGAGCTGA TGGTTTTAAA TCTGGTTTTA AATCTCAAAA AAAAAAAAAA 1910 667 amino acids amino acid single linear unknown 9 Lys Pro Ala Cys Ser Ser Val Arg Leu Thr Thr Arg Glu Lys Pro His 1 5 10 15 Cys Val Cys Gln Asp Pro Met Thr Cys Pro Pro Ala Lys Leu Leu Asp 20 25 30 Gln Val Cys Gly Thr Asp Asn Gln Thr Tyr Thr Ser Ser Cys Tyr Leu 35 40 45 Phe Ala Thr Lys Cys Lys Leu Glu Gly Thr Lys Lys Gly His Gln Leu 50 55 60 Gln Leu Ile Thr Trp Ser Leu Gln Ile Tyr Pro Ala Cys Thr Asp Phe 65 70 75 80 Glu Val Thr Gln Phe Pro Leu Arg Met Arg Asp Trp Leu Lys Asn Ile 85 90 95 Ser Cys Ser Cys Met Asn Leu Thr Leu Asn Thr Leu Asp Ile Ser Thr 100 105 110 Arg Ser Arg Glu Thr Lys Ser Arg Lys Ser Thr Trp Met Lys Lys Arg 115 120 125 Leu Leu Ala Gly Asp His Pro Ile Asp Leu Leu Leu Arg Glu His Arg 130 135 140 Ala Thr Ser Arg Phe Gly Gln Val Arg Gln Leu Leu Thr Cys Arg Gly 145 150 155 160 Gly Ala Gly Cys Arg Gly Ala Arg Ser Gly Ala Gln Gly Cys Gly Gly 165 170 175 Asn Gly Ala Glu Arg Leu Gly Arg Pro Gly Gln Ala Ser Gly Ala Glu 180 185 190 Ala Ser Val Ser Arg Arg Arg Thr Cys Ala Phe Asp Thr Leu Glu Ala 195 200 205 Phe Leu Ile Met Asp Gly Glu Asp Ile Pro Asp Phe Ser Ser Leu Lys 210 215 220 Glu Glu Thr Ala Tyr Trp Lys Glu Leu Ser Leu Lys Tyr Lys Gln Ser 225 230 235 240 Ser Arg Lys Ala Arg Asp Glu Leu Val Glu Phe Gln Glu Gly Ser Arg 245 250 255 Glu Leu Glu Ala Glu Leu Glu Ala Gln Leu Val Gln Ala Glu Gln Arg 260 265 270 Asn Arg Asp Leu Gln Ala Asp Asn Gln Arg Leu Lys Tyr Glu Val Glu 275 280 285 Ala Leu Lys Glu Lys Leu Glu His Gln Tyr Ala Gln Ser Tyr Lys Gln 290 295 300 Val Ser Val Leu Glu Asp Asp Leu Ser Gln Thr Arg Ala Ile Lys Glu 305 310 315 320 Gln Leu His Ser Thr Cys Arg Glu Leu Glu Gln Ala Asn Asp Asp Leu 325 330 335 Glu Arg Ala Lys Arg Ala Thr Ile Val Ser Leu Glu Thr Leu Thr Lys 340 345 350 Leu Asn Gln Ala Ile Glu Arg Asn Ala Phe Leu Glu Ser Glu Leu Asp 355 360 365 Glu Lys Glu Ser Leu Leu Val Ser Val Gln Arg Leu Lys Asp Glu Ala 370 375 380 Arg Asp Leu Arg Gln Glu Leu Ala Val Arg Glu Arg Gln Gln Glu Val 385 390 395 400 Thr Arg Lys Ser Ala Pro Ser Ser Pro Thr Leu Asp Cys Glu Lys Met 405 410 415 Asp Ser Ala Val Gln Ala Ser Leu Ser Leu Pro Ala Thr Pro Val Gly 420 425 430 Lys Gly Thr Glu Asn Ser Phe Pro Ser Pro Lys Ala Ile Pro Asn Gly 435 440 445 Phe Gly Thr Ser Pro Leu Thr Pro Ser Ala Arg Ile Ser Ala Leu Asn 450 455 460 Ile Val Gly Asp Leu Leu Arg Lys Val Gly Ala Leu Glu Ser Lys Leu 465 470 475 480 Ala Ala Cys Arg Asn Phe Ala Lys Asp Gln Ala Ser Arg Lys Ser Tyr 485 490 495 Ile Ser Gly Asn Val Asn Cys Gly Val Met Asn Ser Asn Gly Thr Lys 500 505 510 Phe Ser Arg Ser Gly His Thr Ser Phe Phe Asp Lys Gly Ala Val Asn 515 520 525 Gly Phe Asp Pro Ala Pro Pro Pro Pro Gly Leu Gly Ser Ser Arg Pro 530 535 540 Leu Ser Ala Pro Gly Met Cys Arg Ser Val Cys Glu Cys Pro Ala Ser 545 550 555 560 Gly Ala Pro Ala Leu Leu Gln Gln Pro Arg Thr Pro Thr Pro His Pro 565 570 575 Ser Val Pro Gly Pro Ala Leu Cys Pro Pro Ser Ala Ser Pro His Gly 580 585 590 Trp Gln Arg Ala Gly Cys Met Gln Trp Arg Cys Phe Gly Pro Ala Gln 595 600 605 Pro Gln Asp Ser Ala Arg Tyr Gln Tyr Trp Leu Phe Ser Leu Leu Ala 610 615 620 Val Val Pro Leu Val Ser His Asp Cys Thr Phe Val Gly His Glu Val 625 630 635 640 Ile His Thr Cys Ile Thr Trp Ser Leu Asp Ala Glu Val Pro Ile Cys 645 650 655 His Pro Cys Leu Ile Ala Pro Ala Leu Leu Tyr 660 665 252 amino acids amino acid single linear unknown 10 Met Ala Gln Leu Cys Gly Leu Arg Arg Ser Arg Ala Phe Leu Ala Leu 1 5 10 15 Leu Gly Ser Leu Leu Leu Ser Gly Val Leu Ala Ala Asp Arg Glu Arg 20 25 30 Ser Ile His Asp Phe Cys Leu Val Ser Lys Val Val Gly Arg Cys Arg 35 40 45 Ala Ser Met Pro Arg Trp Trp Tyr Asn Val Thr Asp Gly Ser Cys Gln 50 55 60 Leu Phe Val Tyr Gly Gly Cys Asp Gly Asn Ser Asn Asn Tyr Leu Thr 65 70 75 80 Lys Glu Glu Cys Leu Lys Lys Cys Ala Thr Val Thr Glu Asn Ala Thr 85 90 95 Gly Asp Leu Ala Thr Ser Arg Asn Ala Ala Asp Ser Ser Val Pro Ser 100 105 110 Ala Pro Arg Arg Gln Asp Ser Glu Asp His Ser Ser Asp Met Phe Asn 115 120 125 Tyr Glu Glu Tyr Cys Thr Ala Asn Ala Val Thr Gly Pro Cys Arg Ala 130 135 140 Ser Phe Pro Arg Trp Tyr Phe Asp Val Glu Arg Asn Ser Cys Asn Asn 145 150 155 160 Phe Ile Tyr Gly Gly Cys Arg Gly Asn Lys Asn Ser Tyr Arg Ser Glu 165 170 175 Glu Ala Cys Met Leu Arg Cys Phe Arg Gln Gln Glu Asn Pro Pro Leu 180 185 190 Pro Leu Gly Ser Lys Val Val Val Leu Ala Gly Leu Phe Val Met Val 195 200 205 Leu Ile Leu Phe Leu Gly Ala Ser Met Val Tyr Leu Ile Arg Val Ala 210 215 220 Arg Arg Asn Gln Glu Arg Ala Leu Arg Thr Val Trp Ser Ser Gly Asp 225 230 235 240 Asp Lys Glu Gln Leu Val Lys Asn Thr Tyr Val Leu 245 250 209 amino acids amino acid single linear unknown 11 Met Ser Asp Glu Glu Ala Arg Gln Ser Gly Gly Ser Ser Gln Ala Gly 1 5 10 15 Val Val Thr Val Ser Asp Val Gln Glu Leu Met Arg Arg Lys Glu Glu 20 25 30 Ile Glu Ala Gln Ile Lys Ala Asn Tyr Asp Val Leu Glu Ser Gln Lys 35 40 45 Gly Ile Gly Met Asn Glu Pro Leu Val Asp Cys Glu Gly Tyr Pro Arg 50 55 60 Ser Asp Val Asp Leu Tyr Gln Val Arg Thr Ala Arg His Asn Ile Ile 65 70 75 80 Cys Leu Gln Asn Asp His Lys Ala Val Met Lys Gln Val Glu Glu Ala 85 90 95 Leu His Gln Leu His Ala Arg Asp Lys Glu Lys Gln Ala Arg Asp Met 100 105 110 Ala Glu Ala His Lys Glu Ala Met Ser Arg Lys Leu Gly Gln Ser Glu 115 120 125 Ser Gln Gly Pro Pro Arg Ala Phe Ala Lys Val Asn Ser Ile Ser Pro 130 135 140 Gly Ser Pro Ala Ser Ile Ala Gly Leu Gln Val Asp Asp Glu Ile Val 145 150 155 160 Glu Phe Gly Ser Val Asn Thr Gln Asn Phe Gln Ser Leu His Asn Ile 165 170 175 Gly Ser Val Val Gln His Ser Glu Gly Ala Leu Ala Pro Thr Ile Leu 180 185 190 Leu Ser Val Ser Met Asn Leu Thr Thr Pro Gly Thr Ser Ser Arg Ser 195 200 205 Pro 470 amino acids amino acid single linear unknown 12 Met Ala Ala Ala Leu Lys Pro Ser Ala Pro Glu Ile Arg Lys Ala Ala 1 5 10 15 Gln Glu Phe Ile Asn Tyr Leu Asn Lys Ala Val Thr Pro Phe His Ala 20 25 30 Thr Gln Glu Val Lys Asp Arg Leu Leu Gln Ala Gly Phe Thr Glu Leu 35 40 45 Pro Glu Ser Gly His Trp Asp Ile Gln Pro Thr Ser Lys Tyr Phe Val 50 55 60 Thr Lys Asn Arg Ser Ala Ile Leu Ala Phe Ala Val Gly Gly Ser Tyr 65 70 75 80 Lys Pro Gly Ser Gly Phe Ser Ile Val Val Gly His Thr Asp Ser Pro 85 90 95 Cys Leu Arg Val Lys Pro Ile Ser His Gln Lys Ser Asp Lys Phe Leu 100 105 110 Gln Val Gly Val Ser Thr Tyr Gly Gly Gly Ile Trp Arg Thr Trp Phe 115 120 125 Asp Arg Asp Leu Ser Val Ala Gly Leu Val Ile Val Lys Asn Gly Glu 130 135 140 Lys Leu Gln His Lys Leu Ile Asp Val Lys Lys Pro Val Leu Phe Ile 145 150 155 160 Pro Asn Leu Ala Ile His Leu Glu Thr Asp Arg Thr Thr Phe Lys Pro 165 170 175 Asn Thr Glu Thr Glu Leu Arg Pro Ile Leu Glu Thr Phe Ala Ala Ala 180 185 190 Gly Ile Asn Ala Pro Gln Lys Pro Glu Ser Thr Gly Phe Ala Asp Pro 195 200 205 Arg Asn Ile Thr Asn Asn His His Pro Gln Phe Leu Gly Leu Ile Ala 210 215 220 Lys Glu Ala Gly Cys Gln Pro Glu Asp Ile Val Asp Leu Asp Leu Tyr 225 230 235 240 Leu Tyr Asp Thr Asn Lys Ala Ala Ile Val Gly Met Glu Asp Glu Phe 245 250 255 Ile Ser Gly Ala Arg Leu Asp Asn Gln Val Gly Thr Tyr Thr Ala Ile 260 265 270 Ser Gly Leu Leu Glu Ser Leu Thr Gly Glu Ser Phe Lys Asn Asp Pro 275 280 285 Gln Ile Arg Ile Ala Ala Cys Phe Asp Asn Glu Glu Val Gly Ser Asp 290 295 300 Ser Ala Met Gly Ala Ser Ser Ser Phe Thr Glu Phe Val Leu Arg Arg 305 310 315 320 Leu Ser Ala Gly Gly Ser Thr Thr Ala Phe Glu Glu Ala Ile Gly Lys 325 330 335 Ser Met Leu Ile Ser Ala Asp Gln Ala His Ala Thr His Pro Asn Tyr 340 345 350 Ser Ala Lys His Glu Glu Asn His Arg Pro Ala Phe His Gly Gly Val 355 360 365 Val Val Lys Val Asn Val Asn Gln Arg Tyr Ala Thr Thr Ser Thr Thr 370 375 380 His Ala Ala Leu Lys Gln Val Ala Phe Glu Ala Gln Val Pro Leu Gln 385 390 395 400 Val Val Val Val Arg Asn Asp Ser Pro Cys Gly Ser Thr Val Gly Pro 405 410 415 Ile Leu Ala Thr Lys Leu Gly Leu Gln Thr Val Asp Val Gly Cys Pro 420 425 430 Gln Leu Ala Met His Ser Ile Arg Glu Phe Ala Asp Thr Ser Ser Ile 435 440 445 Tyr Gln Ala Thr Thr Leu Tyr Ser Thr Phe Tyr Glu Arg Leu Ser Thr 450 455 460 Val Leu Ser Asn Met Gln 465 470 

What is claimed is:
 1. An isolated and purified polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1.
 2. An isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide of claim 1, wherein said variant encodes a polypeptide having protease activity.
 3. A probe comprising the polynucleotide of claim
 1. 4. An isolated and purified polynucltide having a sequence which is completely complementary to the polynucleotide sequence of claim
 1. 5. An isolated and purified polynucleotide comprising a polynucleotide sequence of SEQ ID NO:5.
 6. An isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide of claim 5, wherein said variant encodes a polypeptide having protease activity.
 7. An isolated and purified polynucleotide having a sequence which is completely complementary to the polynucleotide of claim
 5. 8. An expression vector comprising the polynucleotide of claim
 1. 9. A host cell comprising the expression vector of claim
 8. 10. A method for producing a polypeptide, the method comprising the steps of: a) culturing the host cell of claim 9 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
 11. A method for detecting a polynucleotide in a sample, the method comprising the steps of: (a) hybridizing the polynucleotide of claim 4 to at least one of the nucleic acids in the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide encoding the polypeptide in the sample.
 12. The method of claim 11 wherein the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
 13. A method of using a polynucleotide to screen a library of molecules or compounds to identify at least one molecule or compound which specifically binds the polynucleotide, the method comprising: a) combining the polynucleotide of claim 1 with a library of molecules or compounds under conditions to allow specific binding, and b) detecting specific binding, thereby identifying a molecule or compound which specifically binds the polynucleotide.
 14. The method of claim 13 wherein the library is selected from DNA molecules, RNA molecules, artificial chromosome constructions, peptides, and proteins.
 15. A method of using a polynucleotide to purify a molecule or compound which specifically binds the polynucleotide from a sample, the method comprising: a) combining the polynucleotide of claim 1 with a sample under conditions to allow specific binding, and b) detecting specific binding between the polynucleotide and a molecule or compound, c) recovering the bound polynucleotide, and d) separating the polynucleotide from the molecule or compound, thereby obtaining a purified molecule or compound. 